HIV-1 is known to aggravate HCV-related liver disease by accelerating HCV replication through as yet unclear mechanisms. Recent studies indicate that HIV-1 Nef can be transferred from HIV-1 susceptible cells to other uninfected susceptible and even to non-susceptible cells through conduits or exosomes. Further, Nef possesses multiple exacerbating functions including intracellular lipid mediation of HCV replication, complexing with cancer-modulating cellular kinases, and interference with anti-HCV host immune defenses. Accordingly, we have studied the role of Nef in HCV-infected hepatocytes to better understand the pathobiology of co- infection. Our data showed that hepatocytes do not support HIV replication. No evidence of virus replication was observed, even when the HIV-1-transfected hepatocytes were co-cultured with Jurkat T cells, indicating that liver deterioration in the co-infected patient is not due to the replication of HIV-1 in the hepatocytes of the HCV infected host. Instead, HIV-1 Nef protein was found to be transferred from expressing T cells to hepatocytes through conduits, wherein up to 16% of hepatocytes harbor the transferred Nef by co-cultivation with nef-expressing Jurkat cells for 24 hr. Moreover, Nef altered the size and numbers of lipid droplets (LD) and consistently up-regulated HCV replication by 1.5~2.5 fold in the target hepatocytes, which is significant in view of the otherwis indolent baseline replication. Nef also dramatically augmented reactive oxygen species (ROS) production, which can activate signaling molecules, such as MAP kinase, to induce TGF1 expression. Besides, Nef increased intracellular and cell surface expression of scavenger receptor B1 (SR-B1), which is integral for virus entry and cholesterol trafficking in Huh7.5.1, implying that Nef can foster susceptibility of HCV infection in non-HCV infected hepatocytes. Further, Nef enhanced ethanol-mediated up-regulation of HCV replication so as to accelerate hepatocellular carcinoma (HCC). Taken together, these data indicate that HIV-1 Nef is a critical element in accelerating HCV-mediated liver pathogenesis via enhancing HCV replication and coordinating modulation of key intra- and extra-cellular molecules for liver decay. Based on these preliminary findings, we propose three Specific Aims for this project. Success in achieving these goals will clarify the pathobiologic mechanisms of HIV-1-mediated exacerbation of liver decay, leading to more effective tools for prognosis and therapeutics against dual virus hepatic disease.